Recombinant toxins are chimeric proteins in which a cell targeting moiety is fused to a toxin (Pastan et al., Science, 254: 1173-1177 (1991)). If the cell targeting moiety is the Fv portion of an antibody, the molecule is termed a recombinant immunotoxin (Chaudhary et al. Nature, 339: 394-397 (1989)). The toxin moiety is genetically altered so that it cannot bind to the toxin receptor present on most normal cells. Recombinant immunotoxins selectively kill cells which are recognized by the antigen binding domain. Fv fragments are the smallest functional modules of antibodies. When used to construct immunotoxins, Fv fragments are better therapeutic reagents than whole IgGs because their small size facilitates better tumor penetration (Yokota et al., Cancer Res., 52: 3402-3408 (1992)). Initially, Fvs were stabilized by making recombinant molecules in which the Variable Heavy (VH) and Variable Light (VL) domains are connected by a peptide linker so that the antigen binding domain site is regenerated in a single protein (a single chain Fv, or “scFv”) (Bird R., et al., Science, 242:423-426 (1988)). Many Fvs, however, could not be stabilized by this approach.
An alternative approach is to stabilize the Fv by a disulfide bond that is engineered between framework regions of the two Fv domains. The disulfide-bond stabilized Fv (termed a “dsFv”) is fused to the toxin through either of the Fv domains (Brinkmann et al., Proc Natl Acad Sci (USA), 90: 7538-7542 (1993)). One striking difference between scFv immunotoxins and dsFv immunotoxins is that dsFv immunotoxins are more stable. Also, dsFv immunotoxins can often be produced with higher yields than scFv immunotoxins (Reiter et al., Biochem, 33: 5451-5459 (1994)).
During the past several years, a number of recombinant toxins have been made using different antibodies (“Abs”) (Reiter and Pastan, Trends Biotechnol., 16:513 (1998)). Several of these recombinant immunotoxins have now been evaluated in phase I trials in patients with cancer. All the recombinant immunotoxins that have been brought to clinical trials have been shown to reduce the size of human cancer xenografts growing in nude mice and to be relatively well tolerated by mice and monkeys (Reiter and Pastan, supra). In a phase I trial, eight partial responses were observed in patients with hematopoietic malignancies treated with an immunotoxin, called anti-Tac(Fv)-PE38 (also known as LMB-2), directed against the α subunit of the IL-2 receptor. Side effects have been observed, however, that cannot be attributed to targeting IL-2R positive cells. These side effects limit the amount of immunotoxin that can be given to humans.
The toxic side effects of recombinant immunotoxins are of two types. One type of toxicity results from specific targeting of normal cells which display the same antigen (“Ag”) as the cancer cell. The second type of toxicity is nonspecific and usually is characterized by damage to liver cells; this increases the serum levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvate transaminase (Kreitman and Pastan, Semin. Cancer Biol., 6:297 (1995)), although other toxic effects may occur (Kreitnan and Pastan, Adv. Drug Delivery Rev., 31:53 (1998)).
Some attempts have been made to alter the toxicity of immunoconjugates such as immunotoxins. Morgan, Jr. et al., U.S. Pat. No. 5,322,678, states that the charge of antibodies can be modified to alter their uptake by the kidneys and hence affect their serum half-life. It indicates that antibodies with high positive charges at physiological pH, such as antibodies with highly basic isoelectric points (pI), are likely to undergo charge interaction with negatively charged patches in the glomeruli of the kidney and to be rapidly cleared from the circulation. The patent states that acidic shifts can be are made by reacting antibodies or antibody fragments with succinic anhydride to modify lysine residues.
Kim et al., J. Label. Compd. Radiopharm. XL:422-430 (1997) states that anti-Tac disulfide stabilized immunoconjugates acylated with glycolate to lower their pI had decreased renal uptake without altering tumor uptake.
Kobayashi et al., Cancer Res. 59:422-430 (1999) states that glycolated, unconjugated anti-Tac antibody fragments known as Fabs which were more anionic had less renal clearance and higher tumor accumulation.